Chemiluminescence determination of proteases by flow injection using immobilized isoluminol

Abstract

A flow-injection system for protease determination is described. A tripeptide–isoluminol derivative was immobilized on an affinity support, and incorporated into the flow system. Injected enzyme sample solutions (0.025 cm³) catalysed the hydrolysis of some of the derivatives at pH 8.47, and the released isoluminol was detected by the chemiluminescence produced by its cobalt-catalysed oxidation with hydrogen peroxide. Linear calibration results were obtained for α-chymotrypsin (from 5 × 10^–4 to 0.41 mg dm^–3), trypsin (from 4 × 10^–2 to 4 mg dm^–3) and a commercial protease (from 2 × 10^–3 to 4 mg dm^–3). The limits of detection were 2.7 × 10^–4, 4 × 10^–2 and 2 × 10^–3 mg dm^–3, respectively.